The long term goal of the proposed work is to identify and characterize the molecular determinants of intrinsic nd acquired drug resistance in human tumor cells. Previous work from this laboratory defined how intra-cellular signaling regulates expression of the MDR1 gene. These results and evidence that inter- cellular contact influences intra-cellular signaling, provided the basis for the new proposed studies to examine how inter-cellular contact influences intrinsic tumor cell drug sensitivity. The influence of multicellular contact on drug and radiation sensitivity has been studied for more than twenty years, yet little is known about the molecular determinants involved. However, evidence from monolayer culture systems suggests that intrinsic sensitivity may be influenced by a) enhanced cell cycle arrest, b) enhanced repair of DNA damage, or c) decreased cell death through survival factor signaling pathways. To determine if multicellular contact decreases the intrinsic sensitivity of tumor cells through any one of these mechanisms and to define the molecular determinants of the multicellular contact effect, we have used a novel II culture model (macroporous gelatin microcarriers, or CultiSphers). Using this model system we have shown at extensive inter-cellular contact between human tumor cells in CultiSphers is associated with decreased sensitivity to radiation (Rasey, et al, 1996), cisplatin (CP) and 4-hydropernxycyclo-phosphamide (4-HC) but not 5-fluorouracil, relative to cells grown in monolayers on gelatin. We use the term contact-induced drug resistance or CIDR to describe this phenotype. CIDR in cells containing wild type p53 is concomitant with 1) equivalent accumulation of CP and formation of platinated DNA (Pt-DNA) adducts in CultSphers relative to monolayers, and 2) accumulation of cells in CultiSphers at the G1 checkpoint following a G2 delay. Preliminary data indicate that CIDR may be p53-independent. We hypothesize that CIDR is mediated by inter-cellular contact-induced signals that regulate intra- cellular NA damage recognition and response pathways. Specific Aim 1 is to determine if CIDR is dependent on P-induced cell cycle arrest. Specific Aim 2 is to determine if CIDR is dependent on repair of Pt-DNA adducts. Specific Aim 3 is to determine if signaling through src, ras and raf kinases influences CIDR. Our experimental system provides a unique tool for investigating the molecular mechanism of CIDR in isogenic cell airs which differ only in growth mode. The results of the proposed studies will provide novel insights into how multicellular growth influences the response of tumor cells to drug treatment and may provide insights applicable o increasing drug efficacy.